杆状病毒转移载体的构建及绿色荧光蛋白在斜纹夜蛾幼虫中的表达

为构建斜纹夜蛾核型多角体病毒 （SpltMNPV）的重组病毒，以该病毒日本C3株基因组DNA为PCR扩增模板，根据GenBank SpltMNPV中国G2株基因序列，设计了两对引物分别扩增多角体蛋白基因的5′端侧翼序列（含启动子）和3′端侧翼序列（含终止子），将这两个片段依次克隆于pUC18质粒载体后，再将绿色荧光蛋白(GFP)基因亚克隆到上述载体的多角体蛋白基因启动子和终止子之间，获得转移载体pSplt-gfp。将pSplt-gfp与野生型SpltMNPV 基因组DNA共转染Spli细胞，通过同源重组和有限稀释法筛选，获得了以gfp基因替代多角体蛋白基因的重组病毒SpltMNPV-gfp。SpltMNPV-gfp感染Spli细胞和斜纹夜蛾幼虫，分别在感染24h和48h后可发现绿色荧光蛋白的表达。该重组病毒的获得，为建立斜纹夜蛾核型多角体病毒表达体系奠定了基础。

Construction of a Baculovirus Transfer Vector and Expression of Baculovirus-mediated gfp Gene in Larvae of Spodoptera litura

To construct a novel baculovirus expression system of Spodoptera litura multicapsid nucleopolyhedrovirus, the 5' end and 3' end-flanking fragments of ph gene were amplified from the genome DNA of SpltMNPV, Japan-C3 strain using two pairs of primers synthesized according to SpltMNPV China-G2 strain genome DNA sequence published in GenBank. To obtain the transfer vector pSplt-gfp, the fragment of gfp gene was inserted into this vector between two fragments tandem linked into pUC18. The spli cells were cotransfected with pSplt-gfp and the wild SpltMNPV genome DNA. The recombinant virus containing gfp was selected with the limited dilution method. The fluorescence can be observed in the spli cells and the 3rd instar larvae after 24 and 48 hours by infection of the recombinant virus, respectively. The result showed that the recombinant virus was obtained successfully. It will be helpful to establish Spodoptera litura multicapsid nucleopolyhedrovirus expression system and more effective pesticide for Spodoptera litura.